PI3K/AKT/mTOR signaling is critical for the cancer initiation and progression. Aberrant PI3K/AKT/mTOR hyperactivation has been documented in a large proportion of breast cancer patients. However, PI3K/AKT inhibitors have shown limited efficacy in the clinic, due to dose-limiting toxicities and emergence of resistance. Thus, identification of aberrant mechanisms of upstream regulation of AKT and identification of downstream mechanisms of PI3K/AKT signal relay to phenotypes associated with malignancy, remains critical. Epigenetic regulation plays an important role in tumorigenesis, and inhibitors targeting epigenetic factors are in clinical trials. Methylation of histones as well as non-histone proteins has been shown to play a functionally pivotal role in human cancers, including breast cancer. However, whether oncogenic signaling pathways, including PI3K/AKT/mTOR, are subject to methylation-dependent regulation has not been explored. Our preliminary data show that AKT undergoes lysyl methylation, a novel mode of regulation that contributes to protein kinase activation in breast cancer. Depletion of the histone methyltransferase SETDB1 reduces AKT activity, suggesting that SETDB1 could be a novel therapeutic target for PI3K/AKT-driven breast cancers. Therefore, in Aim 1 we propose that aberrant expression of SETDB1 in breast cancer contributes to hyperactivation of AKT in a methylation-dependent manner. We will define mechanistically how SETDB1 functions as a novel upstream regulatory mechanism that promotes AKT activation. We will further examine whether genetic ablation of SETDB1 suppresses proliferation in vitro and in vivo. Gene transcription, protein translation and metabolic reprogramming are known to mediate PI3K/AKT/mTOR signaling in cancer. Our preliminary studies have uncovered a previously unrecognized mechanism, whereby the N-glycosyl transferase ALG3 (asparagine-linked glycosylation 3 homolog), is co- amplified with PIK3CA in breast tumors, tightly correlates with a proliferative gene signature in breast cancers and is phosphorylated downstream of PI3K/AKT/mTOR. Deregulation of ALG3 induces ER stress leading to activation of the unfolded protein response (UPR). Thus, in Aim 2, we propose that ALG3 is a functional target of PI3K/AKT/mTOR/S6K1 signaling, and that hyperactivation of this pathway is required to meet the demands of increased protein translation, thereby reducing ER stress. We will determine the mechanism by which PI3K/mTOR signaling regulates ALG3 function and perform functional glycomics in vitro and in vivo. We will determine the contribution of ALG3 to growth in pathway-mutant cells and use combination therapy approaches with PI3K/AKT/mTOR inhibitors and drugs that block ER stress/UPR. The proposed studies will provide the molecular basis and rationale for developing more effective targeted therapies by suppressing the PI3K/AKT pathway based on individual patients? signaling signatures to achieve better treatment outcome.